Immunofluorescent staining of ISH-stained tissues and paraffin sections was performed as described previously. Primary antibodies were diluted as follows: 1:200 rabbit anti-CDH1 (3195, Cell Signaling Technology, Beverly, MA), 1:250 mouse anti-CDH1 (610181, BD Transduction Laboratories, San Jose, CA), 1:100 rabbit anti-phosphoSMAD1/5/8 (9511, Cell Signaling Technology, Danvers, MA). Secondary antibodies were diluted as follows: 1:250 Dylight 549-conjugated goat anti-rabbit IgG (111-507-003, Jackson ImmunoResearch, West Grove, PA), and 1:250 Dylight 488-conjugated goat anti-mouse IgG (115-487-003, Jackson ImmunoResearch). Immunofluorescently labeled tissues were counterstained with 4’,6-diamidino-2-phenylindole, dilactate (DAPI), and mounted in anti-fade medium (phosphate buffered saline containing 80% glycerol and 0.2% n-propyl gallate). Whole mount immunohistochemistry was performed as described previously. Primary antibody was diluted 1:750 rabbit anti-CDH1 and secondary antibody was diluted 1:500 biotin conjugated goat anti-rabbit IgG (BA-1000, Vector, Burlingame, CA).
Dylight 488 conjugated goat anti mouse igg
Manufactured by Jackson ImmunoResearch
Sourced in Panama
DyLight 488-conjugated goat anti-mouse IgG is a secondary antibody that targets mouse immunoglobulin G (IgG) and is conjugated to the DyLight 488 fluorescent dye. This product is designed for use in various immunodetection techniques, such as immunohistochemistry, immunocytochemistry, and Western blotting, where the detection of mouse IgG is required.
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Lab products found in correlation
Mouse anti cdh1, by BD (3 mentions)
Rabbit anti cdh1, by Cell Signaling Technology (2 mentions)
Dylight 549 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Biotin conjugated goat anti rabbit igg ba 1000, by Vector Laboratories (2 mentions)
Dylight 594 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Rabbit anti phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Metamorph version 4, by Molecular Devices (1 mentions)
Dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Rabbit anti dnmt1, by Cell Signaling Technology (1 mentions)
Bz x700, by Keyence (1 mentions)
Dylight 488 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
6 protocols using dylight 488 conjugated goat anti mouse igg
1
Immunofluorescent Staining of ISH-Stained Tissues
2
Immunohistochemical and Fluorescence Staining
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For immunohistochemical staining, tissue sections were incubated with antibodies against superoxide dismutase 2 (Abcam) for 2 hours. The staining was detected using the streptavidin–biotin peroxidase complex method with the DAB Peroxidase Substrate Kit (SK-4100; Vector Laboratories, Burlingame, CA, USA), and counterstained with hematoxylin. For fluorescence staining, frozen-section tissue slides were fixed and blocked, and then slides were triple stained with: mouse antibody against human beta-2-microglobulin (hβ2M; Abcam) followed by DyLight 488-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Sacramento, CA, USA); rabbit antibody against human IgG (Abcam), human/rat sex determining region Y-box 9 (Sox-9; Abcam), or human/rat P450scc (Abcam) followed by DyLight 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) at room temperature for 30 minutes; and 4,6-diamidino-2-phenylindole (Santa Cruz) for the nucleus. All samples were assessed under a fluorescence microscope (Leica Microsystem, Wetzlar, Germany). Images were acquired using MetaMorph version 4.6 (Molecular Devices, Sunnyvale, CA, USA).
3
Immunohistochemical Analysis of Mouse Embryos
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Mouse embryos and fetuses at indicated time points were fixed in 4% paraformaldehyde overnight and dehydrated through a graded series into 100% methanol for storage at −20 °C. Embryos and fetuses were rehydrated, and immunohistochemistry was performed as previously described (73 (link)). The following primary and secondary antibodies were used: 1:50 rabbit anti-DNMT1 (#5032, Cell Signaling Technologies), 1:200 mouse anti-CDH1 (BD 610181), 1:250 DyLight 488-conjugated goat anti-mouse IgG, and 1:250 DyLight 594-conjugated goat anti-rabbit IgG (#35502 and #35560, Jackson ImmunoResearch). Sections were imaged using a Keyence BZ-X700 (Keyence) fluorescence microscope.
4
Immunofluorescent Staining of ISH-Stained Tissues
5
Polyclonal Antibody Production Protocol
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I chose the SVTVREVGDLFQEWLQGNVN sequence for making antisera, as this sequence is present in multiple copies on the precursor (Fig. 1 ). Two milligrams (purity 84%, Proteogenix, Schiltigheim, France) were conjugated to 5 mg of bovine serum albumin using difluorodinitrobenzeze as the conjugation reagent as documented by Tager (1976) (link). Polyclonal mouse antisera were raised in three 6-week-old NMRI female mice as described previously (Veenstra & Ida, 2014 (link)). Tissues were fixed for 1–2 h at room temperature. All other immunohistological procedures are the same as described (Veenstra & Ida, 2014 (link)). Primary antiserum was diluted 1:2,000, the secondary antiserum, DyLight-488-conjugated goat anti-mouse IgG that was from Jackson ImmunoResearch Europe (Newmarket, Suffolk, UK), 1:1,000.
6
Oxidative Stress-Induced Cellular Changes
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Cells grown on coverslips were treated with 0.5 or 1 mM H2O2 for 15 min and then recovered in drug-free medium for the indicated time. Cells were extracted with CSK buffer containing 0.5% Triton X-100, a protease inhibitor cocktail, 10 mM NaF, 10 mM β-glycerophophate and 1 mM DTT for 2 min on ice followed by methanol/acetone fixation at 4°C for 20 min. After blocking in 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min at RT, cells were incubated with primary antibodies diluted in 1% BSA/PBS overnight at 4°C and the next day with secondary DyLight™488-conjugated goat anti-mouse IgG, TRITC-conjugated goat anti-rabbit or mouse IgG antibodies (Jackson ImmunoResearch). Images were obtained using a Carl Zeiss LSM510 confocal microscope.
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